Fig. 1.

T cells are found in abundance within the olfactory neuroblastoma tumor immune microenvironment, most often within the stroma. (A) Representative photomicrographs at 20x magnification from a high Hyams grade ONB of merged and single-color immunofluorescence images assessing the presence of T cells with a validated panel of six biomarkers, CD8 (yellow, Opal 570), CD4 (green, Opal 520), FOXP3 (turquoise, Opal 480) indicated by yellow arrows, Ki67 (red, Opal 690), PD-1 (orange, Opal 620), and synaptophysin (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. Expression of CD4 without CD8 and FOXP3 identified CD4⁺ T helper cells, while expression of CD8 without CD4 and FOXP3 identified CD8⁺ cytotoxic effector T cells. Co-localization of CD4 and FOXP3 without CD8 identified T_regs. T cells with positive Ki-67 nuclear expression were proliferating, and PD-1 positivity denoted PD-1⁺ T cells. Expression of synaptophysin identified tumor cells. (B) Quantification of CD4⁺ T cell, CD8⁺ T cell, and T_reg cell density (cells per mm²), as well as proliferating and PD-1⁺ subcategories of these are compared between ONB tumor and stroma (n = 44 tumor cores). (C) Quantification of tumor specific Ki-67⁺ CD4⁺ T helper cells compared by Hyams Grade. (D) Quantification of tumor specific CD4⁺ T helper cells when comparing patients who presented with and without dural infiltration. For paired comparisons, Wilcoxon matched-pairs signed rank test and for unpaired comparisons, Mann-Whitney U tests were used to test for statistical significance. All lines are graphed to indicate the median value. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns, non-significant